PROJECT SUMMARY Poor packaging of fat in adipocytes is associated with high lipolysis, lipotoxicity and insulin resistance. Lipid droplets (LDs) in adipocytes are distinct intracellular organelles that are the primary storage medium for FFAs as triglycerides (TGs). There is a lack of knowledge of the molecular components of LDs which determine their morphology and TG storing capacity in adipocytes. Thus, there is an urgent need to understand the pathways involved in regulating LDs and TG accumulation in adipocytes. We and others recently identified that fat specific protein (FSP27, also called CIDEC), is associated with LDs and plays a role in fat metabolism in adipocytes. Expressing FSP27 in cells causes increased TG accumulation and larger LDs whereas its depletion results in fragmentation of LDs and increased TG hydrolysis. We also identified a mutation in FSP27 which causes partial lipodystrophy and insulin resistance (IR) in humans. In another study, we found that FSP27 expression was higher in WAT of insulin sensitive vs. insulin resistant obese humans. Our studies show that FSP27 is a major new modulator of LD morphology and function. Based upon our strong preliminary data, we hypothesize that in human adipocytes FSP27 is a critical regulator of LD morphology and is required for efficient TG storage. We further hypothesize that FSP27 regulates TG storage by decreasing basal lipolysis in adipocytes. Together, these steps will protect adipocytes from FFA mediated IR by sequestering FFAs as TGs in LDs. We propose a detailed mechanistic study of structure-function relationships by which FSP27 acts as a multifunctional regulator of LD assembly (Aim 1), TG storage (Aim 2) and insulin sensitivity (Aim 3) in human adipocytes. Our studies will advance the field by identifying the molecular components of LDs that determine their morphology, and elucidating the mechanism(s) involved in TG turnover in adipocytes. Furthermore, these studies will provide tools by identifying the specific amino acid sequences that could be used as therapeutics for insulin resistance.